iTRAQ and RNA-Seq applied in the evaluation of antibiotic stress in multidrug resistant vanB1 Enterococcus faecalis strain. Authors: L. Pinto, C. Torres, C. Gil, J. A. Reales-Calderón, V. Borges, J. P. Gomes, C. Silva, L. Vieira, P. Poeta, G. Igrejas

The collection of vancomycin-resistant enterococci (VRE) isolates of patients from a Spanish hospital revealed the existence of multidrug-resistant strains. Among them was identified vanB1 Enterococcus faecalis strain C2280 colonized in a cardiac valve, showing extensive genotypic and phenotypic concerning antibiotic resistance. For the purpose of understanding the mechanisms and processing of antibiotic resistance we have submitted this strain to different sub-inhibitory states of antibiotic stress of vancomycin and gentamicin, both individually and combined. Isobaric mass tags (iTRAQ)-labeled stressed cells were then compared against a pooled normal control and separated using high pH reverse phase chromatography and on-line reverse chromatography. For peak analysis and quantification was used Proteome Discoverer and MASCOT search algorithm for protein identification. Whole genome sequencing and total RNA-SEQ were conducted for all sub-inhibitory states, allowing the comparison of both genes and transcripts expression. The application of proteomic tools, namely iTRAQ, correlated to transcriptomic tools such as RNA-SEQ, is a valuable resource for the research of sub-inhibitory concentration effects on the general expression of clinical multi-resistant bacteria. Accurate identification and quantification of genes, transcripts and proteins supply an exact knowledge of how the pathways behave facing a given stress, which is very important when studying a public health concern like multidrug-resistant bacteria that reveal high adaptive and survival capacities.